PROJECT SUMMARY/ABSTRACT Members of the Candida genus of fungi form part of the normal human microbiota but are also opportunistic pathogens capable of causing serious mucosal and systemic infections. Candida cells grow and divide in suspension (planktonic) cultures, but they also form resilient and drug resistant biofilms ? organized, tightly- packed communities of cells attached to a surface. Biofilms colonize many niches of the human body and can also form on implanted medical devices, where they are a major source of new infections in patients. Mortality rates from Candida infections are particularly high in immunocompromised individuals, where life-threatening colonization and invasion of parenchymal organs can occur once the infection has disseminated through the bloodstream. Because (1) the mortality rate of disseminated infections is high (approximately 50 percent), (2) biofilms are a major source of these infections, and (3) biofilms are also resistant to current antifungal drugs, rapid and early detection of biofilm formation is critical for improving disease outcome. The Craik laboratory at UCSF (collaborators on this proposal) recently developed a novel mass spectrometry-based screening technology to identify the global substrate specificity and kinetic efficiency of proteases in complex biological mixtures. This technology, referred to as Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), allows for unbiased and simultaneous detection of all protease activities in a given sample; it employs a library of rationally designed peptide substrates and monitors their cleavage. In consultation with the Craik laboratory, we have applied this global profiling strategy to identify biofilm-specific, planktonic-specific, and broad- spectrum protease activities with the goal of developing protease-cleavable fluorogenic substrates that will enable the rapid and sensitive enzymatic detection of biofilm and disseminated infections from a broad range of Candida species. Through this profiling of the C. albicans secreted proteome, we have identified two secreted aspartyl proteases, Sap5 and Sap6, and a subtilisin-like serine protease, Kex2, that are produced by C. albicans biofilms. We have developed first-generation fluorogenic substrates specific for each activity; genetic experiments (using C. albicans strains deleted for a given protease) have confirmed that these substrates show high specificity for their target protease. We have also shown that these same substrates can detect proteolytic activity from other pathogenic Candida species. Finally, we have demonstrated that the Sap6-cleavable fluorogenic substrate can detect infection-specific activity in serum isolated from rats that have an implanted catheter infected with a C. albicans biofilm. Based on these preliminary results, in Phase I of this R43 proposal, we propose to develop a protease-profiling pipeline for the discovery of additional proteases secreted from C. albicans and other pathogenic Candida species. Optimized protease-cleavable fluorogenic substrates will be continually developed, refined, and tested for their ability to accurately detect Candida biofilms and planktonic cells grown in vitro (Aim 1) and in vivo using a preclinical murine catheter biofilm model and a murine disseminated infection model (Aim 2). In Phase II of this award, we will translate these discoveries to humans by evaluating the clinical efficacy of our protease activity-based approach for Candida biofilm and disseminated infection detection. The eventual goal is the development of an optimized substrate kit for the rapid diagnosis of biofilm-associated and disseminated infections.